D-Malic enzyme of Pseudomonas fluorescens.
By the enrichment culture technique 14 gram-negative bacteria and two yeast strains were isolated that used D(+)-malic acid as sole carbon source. The bacteria were identified as Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas aeruginosa and Klebsiella aerogenes. In cell-free extracts of P. fluorescens and P. putida the presence of malate dehydrogenase, D-malic enzyme (NAD-dependent) and L-malic enzyme (NADP-dependent) was demonstrated. D-Malic enzyme from P. fluorescens was purified. Stabilization of the enzyme by 50 mM ammonium sulphate an 1 mM EDTA was essential. Preparation of D-malic enzyme that gave one band with disc gel electrophoresis showed a specific activity of 4-5 U/mg. D-Malic enzyme requires divalent cations. The Km values were for malate Km = 0.3 mM and for NAD Km = 0.08 mM. The pH optimum for the reaction was found to be in the range of pH 8.1 to pH 8.8. D-Malic enzyme is partially inhibited by oxaloacetic acid, meso-tartaric acid, D-lactic acid and ATP. Determined by gel filtration and gradient gel electrophoresis, the molecular weight was approximately 175 000.
D002474:Cell-Free System; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology; D008291:Malate Dehydrogenase / Q000302:isolation & purification*; D008970:Molecular Weight; D011549:Pseudomonas / Q000201:enzymology; D011551:Pseudomonas fluorescens / Q000201:enzymology*
D008291:Malate Dehydrogenase; C034544:D-malate dehydrogenase (decarboxylating)